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Surface Plasmon Resonance: antigen-antibody interactions. Vamsi K. Mudhivarthi. Analytical Techniques for Biomaterial Characterization. Scanning Probe Microscopy (SPM): image changes at surface Attenuated Total Reflectance Infrared Spectroscopy (ATR-IR):

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Presentation Transcript
analytical techniques for biomaterial characterization
Analytical Techniques for Biomaterial Characterization
  • Scanning Probe Microscopy (SPM):
    • image changes at surface
  • Attenuated Total Reflectance Infrared Spectroscopy (ATR-IR):
    • study conformational changes at solid-aqueous interfaces, although lacks sensitivity
  • Spectral Ellipsometry:
    • determination of thickness and refractive index of adsorbed layer
  • Surface Plasmon Resonance (SPR):
    • rapidly monitor dynamic processes to a wide range of biomedically relevant interfaces.
what is spr
What is SPR?
  • Surface Plasmon:Longitudinal charge density wave along the interface of two media, where one is metal and other is dielectric
spr principle
SPR Principle
  • Linear relationship is found between resonance energy and mass concentration of biochemically relevant molecules.
advantage of using gold film
Advantage of using gold film
  • Gold:Non-magnetic, surface plasmon wave is p-polarized, and due to its electromagnetic and surface propagating nature, creates enhanced evanescent wave
spr kinetics of association phase
SPR: Kinetics of Association phase
  • C= Concentration of analyte
  • Rmax = maximum analyte binding capacity of the surface in RU
  • R = SPR signal at time t in RU

or

spr kinetics of association phase9
SPR: Kinetics of Association phase
  • Kd is not very reliable as KaC >>Kd
spr kinetics of dissociation phase
SPR: Kinetics of Dissociation phase
  • Rt is response at time t in RU
  • R0 is response at an arbitrary starting point

After integration and logarithm

we have

applications of spr
Applications of SPR
  • Physical applications: measure dielectric properties, adsorption processes, surface degradation or hydration of
    • Thin organic monolayers or bilayers
    • Polymer films
  • Biological applications: as biosensors for specific biological interactions including adsorption and desorption kinetics, antigen-antibody binding and epitope mapping for determination of
    • Biomolecular structure and interactions of proteins, DNA & Viruses
    • Lipid Bilayers
    • Non-specific biomolecular interactions-bio-compatibility
    • Tissue engineering
spr physical applications
SPR: Physical applications

Thin organic monolayers or bilayers

Polymer films

spr biological applications
SPR: Biological applications

Tissue engineering

Epitope mapping

single step analysis fmdv antigen antibody interactions
Single step analysis: FMDV antigen-antibody interactions

Ka = 9.0 * 104 M-1 S-1

Kd = 1.2 * 10-3 S-1

advantage of spr
Advantage of SPR
  • Ability to perform real-time measurement:
    • Insight to dynamic nature of binding system and layer formation
  • Use of selective slides to study binding events:
    • Eliminate the need for labeled reactants
  • Exceptional sensitivity:
    • Small quantities of purified reagents are required
disadavantages
Disadavantages
  • Disadvantage of SPR:
    • Lack of sensitivity when monitoring low molecular weight adsorbates
    • Rate limiting factor of mass transport-affecting kinetic analysis
  • Methods to improve sensitivity:
    • Coupling to AFM
    • Coupling with Mass-spectrometry
references
References
  • Green R. J., Frazier R. A., et. al., Biomaterials21 (2000) 1823-1835.
  • http://brahms.chem.uic.edu/~cgpage/research/sprintro.html
  • Biacore Manual
  • David Andreu et. al., Journal of immunological methods235 (2000) 101-111.
  • A. McGill et. al., Journal of immunological methods297 (2005) 143-152.
  • Marc H. V. Van Regnmortel et. al., Journal of molecular recognition11 (1998) 163-167.
  • Daniele Altschuh et. al., Biochemistry31 (1992) 6298-6304.